The invention relates to the field of separation of charged molecules, and in particular the separation of mixtures of charged molecules.
Complex charged molecule mixtures, such as protein mixtures can be separated by isoelectric focusing under denaturing conditions in gel tubes or strips that contain either soluble ampholytes (Klose, (1975) Humangenetik 26, 231-243; O""Farrell, (1975) J. Biol. Chem. 250, 4007-4021; Scheele, (1975) J. Biol. Chem. 250, 5375-5385) or immobilines (Bjellqvist et al. (1982) J. Biochem. Biophys. Meth. 6, 317-339). For quantitative comparisons in changes in total protein profiles a second dimension separation can be done on a conventional SDS polyacrylamide gel electrophoresis (PAGE) slab gel.
Current two dimensional (2D) methods, however, lack both adequate resolution and sufficient dynamic range for resolving and detecting large numbers of charged molecules, for example, the protein components present in eukaryotic proteomes which can comprise over 10,000 proteins. A major disadvantage of existing 2D gel methods when applied to a large number of charged molecules is that the maximum sample loading capacity is fairly low, which results in detection of only the most abundant charged molecules when currently available stains are used (Herbert et al. 1997; Williams 1999; Quadroni et al. 1999). Increasing the amount of sample above an optimal level results in horizontal streaking of many proteins. Although current IPG-based 2D gels have much higher resolution than alternative separation methods, not all charged molecules in a sample can be resolved by a single IPG gel. This incomplete resolution contributes to errors in subsequent quantitation and identification of charged molecules. Hence, effective analyses of complex charged molecule mixtures such as extracts from eukaryotic cells or tissues require improved separation methods capable of resolving and quantitatively detecting thousands of components.
One method for resolving a large number of charged molecules is prefractionation of sample proteins prior to further analysis. Previously reported prefractionation methods prior to 2D PAGE include sequential extractions with increasingly stronger solubilization solutions (Molloy et al. (1998) Electrophoresis 19, 837-844), subcellular fractionation (Huber et al. (1996) Electrophoresis 17, 1734-1740) and selective removal of the most abundant components (Lollo et al. (1999) Electrophoresis 20, 854-859). Other alternatives include conventional chromatography techniques, such as gel filtration, ion exchange, or affinity chromatography. The use of these methods, however, can result in an incomplete separation of charged molecules between fractions and a poor yield. Using current methods cross contamination of specific charged molecules between fractionated pools can seriously complicate quantitative analyses and comparisons, since many charged molecules appear in more than one fraction and the degree of cross contamination is often highly variable.
Preparative isoelectric focusing as a protein prefractionation procedure was proposed by Bier et al. (in: Peptides: Structure and Biological Functions (Gross and Meienhofer, eds., pp.79-89, Pierce Chemical Co., Rockford, Ill., 1979) and a commercial version called Rotofor(trademark) was produced by Bio-Rad (Hercules, Calif., USA). It is built as a rotating chamber divided into 20 compartments and uses solution isoelectric focusing to fractionate samples. However, this apparatus has no separation barriers and is typically a low resolution technique with relatively large volumes for individual fractions. Righetti et al. ((1989) J. Chromatogr. 475, 293-309) described a multi-compartment electrolyser in which each compartment is separated by a polyacrylamide gel membrane with a specific pH produced by immobilines that are incorporated into the polyacrylamide membranes. A commercial apparatus, called IsoPrime(trademark), incorporating this principle has been marketed (Hoefer Pharmacia, San Francisco, Calif.). The IsoPrime(trademark) unit has been developed primarily for large scale purification of individual proteins starting with partially purified preparations, not for fractionation of crude extracts. The unit has large separation chambers connected to peristaltic pumps and external chambers to further expand the volumes of individual fractions (about 30 ml). While the IsoPrime(trademark) unit can provide high quality separations, its large volume and design make it impractical for prefractionation of samples containing complex mixtures of charged molecules, especially under denaturing conditions. Similarly, other preparative isoelectric focusing instruments suffer from at least several of the limitations encountered with either the Rotofor(trademark) or the IsoPrime(trademark); specifically these instruments: (1) require a large sample volume, (2) produce large volume, dilute fractions that need to be concentrated with attendant losses, (3) exhibit poor resolution, or (4) involve expensive, complex instrumentation.
Better prefractionation methods for the separation of large numbers of charged molecules should improve the detection of minor charged molecules occurring in a mixture and increase the total number of protein components that can be identified (Quadroni and James, (1999) Electrophoresis 20, 664-677; Williams (1999) Electrophoresis 20, 678-688). The ideal prefractionation method would resolve complex mixtures such as total extracts of eukaryotic cells or tissues into a small number of well-separated fractions.
It is an object of the invention to provide a novel device and method for the separation of mixtures of charged molecules. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a chamber for holding a liquid. The chamber has a first porous charged membrane partition or a first membrane permeable to small ions at a first end and a second porous charged membrane partition or a second membrane permeable to small ions at a second end which is opposite the first end. The chamber also has at least one porous charged membrane partition positioned along the chamber to define a plurality of compartments within the chamber such that each compartment holds a volume of liquid less than about 4 ml.
Another embodiment of the invention provides a chamber for holding a liquid. The chamber has a first porous charged membrane partition or a first membrane permeable to small ions at a first end and a second porous charged membrane partition or a second membrane permeable to small ions at a second end which is opposite the first end. The chamber also has means for separating a mixture of at least ten species of charged molecules in liquid.
Yet another embodiment of the invention provides a method of separating a mixture of charged molecules. A mixture of charged molecules in solution is added to a chamber as described above and a direct current is applied between the first end and the second end of the chamber. The charged molecules are separated.
Still another embodiment of the invention provides a method of separating a mixture of at least about 10 species of charged molecules in liquid. The charged molecules are added to a chamber for holding liquid having a first porous charged membrane partition or a first membrane permeable to small ions at a first end and a second porous charged membrane partition or a second membrane permeable to small ions at a second end which is opposite the first end. At least one porous charged membrane partition is positioned along the chamber to define a plurality of compartments within the chamber. A direct current is applied between the first end and the second end of the chamber, whereby the charged molecules are separated.
This invention provides a novel small-scale solution isoelectric focusing device and method that can reproducibly fractionate charged molecules into well-defined pools. This approach can be applied to complex charged molecule samples, such as eukaryotic proteome samples where reproducible resolution and quantitation of greater than 10,000 protein components is feasible.